Patent Application
20110165178
The present invention relates to vitro diagnosis methods of prostate cancer from a test biological sample, comprising measuring the expression level of KIAA0153 in said test biological sample, as well as to methods for screening compounds inhibiting KIAA0153 gene expression or biological activity and uses of such KIAA0153 inhibiting compounds.
Prostate cancer is a public health problem of major and increasing importance particularly as the European population ages. In the European Union, it accounts for over 35,000 deaths every year, associated with considerable morbidity.
Many patients are either over-treated or under-treated, due to the absence of reliable prognostic markers. A usually used marker for diagnosis is PSA (prostate specific antigen). However, most men with an elevated PSA test turn out not to have cancer; only 25 to 30 percent of men who have a biopsy due to elevated PSA levels actually have prostate cancer. The PSA test is thus not specific enough to screen for prostate cancer and new markers that perform better than PSA thus need to be developed.
In addition, there is currently no satisfying treatment for advanced prostate cancer. Indeed, advanced prostate cancer treatment is usually performed by hormone ablation therapy and/or prostatectomy, which is not satisfactory since advanced prostate cancer invariably progresses to hormone refractory disease, leading to death in 3-5 years. There is thus also a need for improved prostate cancer treatments.
The tyrosination cycle has an important role in the cell. In particular, tubulin is subjected to a number of post-translational modifications that regulate the function and organization of microtubules (1), which are essential components of the cell cytoskeleton. One of these modifications is the cyclic removal and re-addition of the C-terminal tyrosine of α-tubulin by tubulin carboxypeptidase and tubulin-tyrosine-ligase (TTL), respectively.
Tubulin tyrosination is conserved among eukaryotes (2) and suppression of TTL in mice causes perinatal death (3). Dividing TTL null cells retain a minor pool of tyrosinated tubulin (Tyr-tubulin), whereas postmitotic TTL null neurons lack Tyr-tubulin and display abnormalities related to neurite growth and axonal differentiation. There is evidence that the tyrosination cycle has a role in proliferation and cancer. Inhibition of TTL accelerates cell proliferation (4). TTL is progressively suppressed during tumor progression in animal models, resulting in accumulation of Glu-tubulin (5). Additionally, TTL is frequently lost in human cancers and tubulin detyrosination is associated with tumor aggressiveness and poor prognosis (6-7).
Interestingly, there are about 14 genes coding for proteins containing TTL domains in the human genome (8) suggesting that they have multiple roles, but the physiological function of most of these proteins and the nature of their substrates are still unknown.
The inventors have performed a phenotypic and functional analysis of one of these novel genes, KIAA0153 (Genbank accession number BC001070). KIAA0153 codes for a 72 KDa protein with a C-terminal TTL domain (
In contrast, the inventors have surprisingly found that KIAA0153 miss-expression interferes with the normal tubulin tyrosination cycle resulting in changes in the levels of Tyr- and Glu-tubulin. Furthermore it induces mitotic aberrations and chromosome instability. KIAA0153 is expressed in the proliferative cell layer of prostate glands and expression increases with prostate cancer progression. Higher expression in Gleason patterns 4 and 5 suggest that KIAA0153 may be a marker of risk of progression and requirement of more aggressive treatment. According to its expression profile, structure and function, KIAA0153 is thus a new marker of prostate cancer and may represent a novel target for prostate cancer therapy.
The invention thus concerns an in vitro diagnosis method of prostate cancer from a test biological sample, comprising measuring the expression level of KIAA0153 in said test biological sample.
In one embodiment, said in vitro diagnosis method further comprises measuring the expression level of KIAA0153 in a healthy control biological sample and comparing the expression level of KIAA0153 in both samples. By “healthy sample” is meant a sample from a subject that is not suffering from prostate cancer.
Preferably, prostate cancer is diagnosed when the ratio of the expression level of KIAA0153 in said test biological sample to the expression level of KIAA0153 in said healthy control biological sample is superior to 1.5, advantageously superior to 1.6, 1.7, 1.8, or 1.9.
The results obtained by the inventors show that KIAA0153 is especially highly expressed in metastatic prostate cancer. In an embodiment of an in vitro diagnosis method according to the invention, said method thus further comprises diagnosing metastactic prostate cancer when the ratio of the expression level of KIAA0153 in said test biological sample to the expression level of KIAA0153 in said healthy control biological sample is superior to 2.0.
The in vitro diagnosis method according to the invention may be performed using various types of test biological samples, including biological fluids such as blood, serum, plasma or urine, as well as biological tissues samples. In a preferred embodiment, said test biological sample is a prostate sample. In particular, said prostate sample may be selected from a prostate biopsy, a prostate resection, or a cell line derived from a prostate biopsy or a prostate resection. In another preferred embodiment, said test or healthy biological sample is a biological fluid sample, in particular a serum sample. Preferably, the test sample and the healthy control sample are the same kind of biological sample to allow for a significant comparison. This way, if the test sample is a prostate sample, then the healthy control sample is also preferably a prostate sample, and if the test sample is a serum sample, then the healthy control sample is also preferably a serum sample.
The measure of KIAA0153 expression level may be performed either at the proteic or at the nucleic level.
In one embodiment, the expression level of KIAA0153 is determined at the protein level. Various technologies well-known in the art may be used to measure the amount of KIAA0153 protein. Examples of suitable technologies to determine the expression level of KIAA0153 at the protein level include immunohistochemistry, immunoblotting, immunofluorescence, or mass spectrometry, but any person skilled in the art will be able to find other appropriate technologies.
In another embodiment, the expression level of KIAA0153 is determined at the nucleic level. In particular, such a measure may be done by measuring the amount of KIAA0153 mRNA or corresponding cDNA. Various technologies well-known in the art may be used to measure the amount of KIAA0153 mRNA or corresponding cDNA. Examples of suitable technologies to determine the expression level of KIAA0153 at the nucleic level include in situ hybridization or quantitative PCR, but any person skilled in the art will be able to find other appropriate technologies.
The results obtained by the inventors show that KIAA0153 may be considered as a new therapeutic target for treating prostate cancer. The invention thus also concerns a method for screening compounds inhibiting KIAA0153 expression, comprising:
A host cell expressing KIAA0153 is a cell in which expression of KIAA0153 is detectable. It may be natural or genetically engineered, however, a genetically engineered host cell which has been transfected (transiently or stably) or transformed with an expression vector capable of directing the expression of KIAA0153 is preferred, since it permits to have a controlled expression level of KIAA0153.
As already described for diagnosis methods according to the invention, the expression level of KIAA0153 may be measured either at the proteic or nucleic level using various technologies.
A test compound will then be considered as inhibiting KIAA0153 expression if KIAA0153 expression is lower in host cells cultured in the presence of said compound than in host cells cultured in the absence of said compound.
The invention also concerns a method for screening compounds inhibiting KIAA0153 biological activity, comprising:
Indeed, KIAA0153 biological activity, which is intended to mean its action in a cell expressing it, may be inhibited either by inhibiting KIAA0153 expression, or by interfering with KIAA0153 action in the cell. The inventors have found that KIAA0153 inhibits TTL tyrosination activity. Thus, a compound inhibiting KIAA0153 biological activity should restore TTL activity.
TTL activity consists in tyrosination of tubulin. Thus, in one embodiment of a method for screening compounds inhibiting KIAA0153 biological activity, TTL activity is measured by measuring incorporation of exogenous tyrosine into tubulin.
This may be done using various technologies easily implemented by a skilled artisan. For instance, the added exogenous tyrosine may be modified to be detectable, in particular by addition of a chemical group that may then be traced, such as a radioactive group, a labelled group (fluorescent, chemiluminescent, etc. . . . ). The measure of tyrosine incorporation may also be done by removing tubulin and microtubules and measuring the remaining tyrosine concentration.
Alternatively, inhibition of TTL activity results in an increased concentration in Glu-tubulin. Thus, in another embodiment of a method for screening compounds inhibiting KIAA0153 biological activity, TTL activity is measured by measuring the amount of Glu-tubulin. This measure is easily performed by well-known technologies.
All sorts of compounds may be screened using the methods of screening according to the invention described above. However, some potential inhibitors of KIAA0153 function immediately appear, including antibodies, siRNAs or antisense nucleic acids directed against KIAA0153.
The invention thus further concerns an antibody directed against KIAA0153 protein. Such an antibody may be easily generated using well-know routine technologies for a skilled artisan. The term “antibody” is intended to mean not only an antibody as such but also antibody fragments such as Fab or Fab′2 or ScFv fragments. It may be polyclonal or monoclonal, and may have been further chimerized or humanized using conventional technologies.
The invention also relates to any antibody directed against KIAA0153 protein as described above, as a medicament. Indeed, as a compound able to interfere with KIAA0153 biological activity, such an antibody may be used as a medicament, in particular for treating prostate cancer.
The invention further concerns the use of a KIAA0153 inhibiting compound for manufacturing a medicament for treating prostate cancer. Any compound able to inhibit KIAA0153 biological activity and/or expression may be used for this purpose. Examples of suitable compounds include compounds selected from an antibody, a siRNA or an antisens nucleic acid directed against KIAA0153.
The invention also concerns a method for treating prostate cancer in a patient in need thereof, comprising the administration of a KIAA0153 inhibiting compound.
Preferably, a treatment comprising a KIAA0153 inhibiting compound is chosen if the ratio of the expression level of KIAA0153 in said test biological sample to the expression level of KIAA0153 in said healthy control biological sample is superior to 1.5, advantageously superior to 1.6, 1.7, 1.8, 1.9 or 2.0.
HEp-2, HCT-116 cells and prostate cell lines were maintained following ATCC (American Type Cell Collection) directions. Transfections were performed by the Calcium-phosphate method. To obtain stable clones, transfected cells were trypsinysed after 48 hours post-transfections (h.p.t.) and splitted into medium containing puromycin (3 mg/ml). The selection medium was replaced every 3 days and clones were isolated after 10-14 days post-transfections (d.p.t.). To obtain double stable transfectants, single stable transfectants (puromycin resistant) were transfected with pBOS H2B-GFP (9) (histone 2B-GFP; BD PharMingen) expression vector and selected with blasticidin (2 mg/ml). Typically, we isolated 5 positive clones by immunofluorescence and western-blotting, per stable transfection. KIAA0153 cDNA clone was obtained from ATCC (MGC-2635; IMAGE: 3504490; Genbank number BC001070; human placenta). For the expression of flag epitope-tagged proteins, the coding region was PCR-amplified using oligos with XhoI and BclI restrictions sites (CGCCTCGAGATGGAGGCCGAGCGGGGT SEQ ID NO:1, and CGCTGATCACTAGACAAGGCAGGTAACG SEQ ID NO:2) and cloned as a fusion protein into the XhoI/BglII window of pSG5-puro-flag expression vector (10), a modified version of the puromycin-resistant vector pSG5-puro. To obtain the antisense version, we used oligos with XhoI sites. The coding region was also amplified with oligos containing BclI sites and cloned into BglII site of pSG5-puro (without the flag epitope). Alternatively, the coding region amplified with oligos containing EcoRI sites was cloned into pGEM-Teasy (Promega) and pcDNA3.1-(Invitrogen) to obtain antisense and sense versions. The antisense version of KIAA0153 is also referred in the figures as Control-2. Although we did not observe significant differences with respect to vector stable transfectants or parental cells in terms of KIAA0153 expression or functional assays, we maintained it in some experiments to serve as negative control. A clone containing human tubulin tyrosine ligase sequences was purchased to ATCC (MGC-46235; IMAGE:5767758; Genbank number BC036819 human brain). The coding region was PCR-amplified with oligos containing the BamHI sites (GATCGGATCCATGTACACCTTCGTGGTACGC SEQ ID NO:3, and CAGGGATCCTCACAGCTTGATGAAGGCA SEQ ID NO:4) and cloned into pSG5-puro-flag expression vector to obtain sense and antisense versions of the gene. Additional vector used were pGFP-C1 (Clontech) for transfection controls, pSuperPuro (Oligoengine) and shRNA153 (shRNA stable down-regulation of KIAA0153 mRNA (mRNA targeted-sequence: GAGUUCAUCCCCGAGUUUG SEQ ID NO:5; oligos were cloned into the pSuperPuro vector). Details on individual plasmids constructs, which were all verified by sequencing, are available upon request. For cell proliferation assay we seeded cells on 96-well plates (3000/well) and use MTT-based colorimetric assay (Chemicon Int.) following manufacturer's directions. When 3-nitrotyrosine (NO2Y Sigma) was used, it was added to cells 24 hours post-transfection at a final concentration of 400 mM. After 48 hours incubation (72 h.p.t) cells were harvested and processed for immunoblotting.
Cells growing in culture plates were rinsed twice in ice-cold PBS, scraped and centrifuged. The cell pellet was lysed in extraction buffer (50 mM Tris-HCl pH 8, 5 mM EDTA pH 8, 150 mM NaCl, 1% Nonidet-P40, 0.02% sodium azide, 1 mM PMSF, 1×PIC (protease inhibitor cocktail, Amersham). Lysates were sonicated at 0° C. for 30 sec and then cleared by centrifugation. Protein concentrations were measured by the Bradford method and 40 mg of total protein were fractionated by 8% SDS-PAGE. Alternatively cell pellets were directly lysated in Laemli buffer, sonicated, boiled and loaded onto the gels. Proteins were transferred to nitrocellulose paper. Western blots were blocked for 1 hour in 5% dried-fat free milk in PBS-Tween 0.05% (TBS-Triton X-100 0.1% for the detection of Glu-tubulin), and then incubated 2 hours with specific antibodies diluted in blocking solution: polyclonal serum against KIAA0153 (1/500), nitrotyrosine (1/1000; Upstate Biotechnology), α-tubulin (1/2000; clone DM1A; Sigma), Tyr-tubulin (1/2000 clone 1A2; Sigma), Glu-tubulin (1/500; Synaptic Systems), β-tubulin (1/2000; Sigma), Tata box protein (TBP) (1/2000; IGBMC monoclonal antibodies facility), Flag (1/2000; IGBMC monoclonal antibodies facility). Blots were washed and incubated with specific secondary antibodies coupled to HRP (Jackson immunoresearch) and enhance chemiluminescence reagents (ECL, Pierce) were used for detection.
Unsynchronized cells growing exponentially were fixed in 70% ethanol (−20° C.), washed and stained with propidium iodide. Detached cell populations were collected and combined with adherent cells for all experiments. FACScalibur cytometer (Becton Dickinson) and CellQuest software were used for acquisition and Modfit software (Verity) for cell cycle analysis.
For time-lapse imaging, double stable transfectants (see cell culture and transfections) were analysed on a DMJRE2 inverted microscope (Nikon) with a ×40 objective enclosed in a temperature- and CO2 incubator. Images were acquired with a CCD camera at 20 min intervals and analysed by the Metamorph software.
We performed standard metaphase spreads and Giemsa staining. Typically, 40 metaphase plates of each preparation were analysed. For KIAA0153 immunofluorescence, cells were grown on chambers slides were fixed in Methanol (−20° C.) for 5 min and 30 sec in Acetone (−20° C.). For the rest of antigens we fixed the cells in Methanol-EGTA 1 mM (−20° C.) for 10 min and then 15 min in 4% paraformaldehyde in PBS. Cells were permebealised by a 5 min wash with PBS-0.1% Triton X-100. Cells were blocked with PBS/BSA 1%, and then incubated with specific antibodies diluted in PBS/BSA 1%: polyclonal antibodies raised against KIAA0153 (1/500), Tyr-tubulin (1/500), Glu-tubulin (1/100), polyclonal CLIP-170 (1/250; gift from Jan De Mey (USBS, Strasbourg), dynein (1/200; Sigma), γ-tubulin (from mouse and rabbit origins; Santa Cruz) followed by secondary antibodies coupled to FITC and Cy3 (Jackson Immunoresearch). Slides were mounted with mounting medium containing DAPI (vectashield; Vector). Images were acquired and analysed by the Metamorph software package (Molecular Devices).
Specimens of local tumor were obtained from untreated patients who underwent radical prostatectomy after tumor diagnosis in a PSA based screening program. In total, 31 benign specimens, 12 PINs, 51 specimens of local tumors, 27 local recurrent tumors (LRT), 8 lymph node and 11 distant metastasis (10 bone, 1 lung) were used. For the analysis of recurrent tumors and metastasis, tissue arrays were employed. A tissue array of locally recurrent tumors was constructed using 2 mm cores from specimens of local resections performed in patients with obstructive symptoms after failure of androgen ablation therapy. These specimens represented “androgen-insensitive” tumors. For the analysis of metastases a lymph node and a distant metastases array was constructed in one paraffin block using three 0.6 mm-cores per case. Immunohistochemistry was performed on 5 m-thick formalin-fixed paraffin-embedded tissue sections using polyclonal antibodies raised against KIAA0153 according to a standard Horseradish peroxidase (HRP) immunohistochemistry protocol. After deparaffination in xylenes and rehydration in decreasing ethanol concentrations, tissue sections were heated in citrate buffer pH 6 in a microwave oven for 30 minutes for antigen retrieval. After washing tissue sections were treated for 10 min with 3% H2O2 in methanol for inactivation of endogenous peroxidase activity, blocked for 1 hour in Tris-buffered saline solution containing 5% skim-milk powder and 0.5% Tween20 (TBSTM5) and incubated over night with antibody diluted 1:200 in TBSTM2 at 4° C. For staining, a broad spectrum HRP DAB kit was employed according to the protocol of the manufacturer (Zymed PicTure PLUS Kit, Broad Spectrum, DAB, Zymed). After final washing steps the sections were counterstained with Gui's hematoxylin solution (Sigma) or methyl green (DAKO). Specificity of staining was controlled by including a control antibody (DAKO Cytomation) and stains without first antibody or pre-immune serum. Immunoreactivity was scored according to the Gleason pattern using a 4 point scaling system: 1, no staining, 2, weak staining, 3, intermediate staining, 4, strong staining. For ISH, 5 m-thick sections were deparaffinized, rehydrated in decreasing concentrations of ethanol and then air-dried. The slides were then washed in PBS three times for 10 min each. The tissue was permeabilized by proteolytic digestion with proteinase K (6 mg/ml in 50 mM Tris-HCl, pH 7.6) at 50° C. for 10 min. The reaction was stopped by rinsing the slides twice in PBS for 5 min each and heating at 92° C. for 2 min in a heat block. The slides were placed in PBS for 5 ml HCI 0.2N for 20 mm, PBS for 5 mm, and subsequently dehydrated through washes in graded ethanols, for 5 min each. The tissue was placed in 2×SSC at 70° C. for 5 min and then at 92° C. for another 5 minutes. Immediately after that, the tissue sections were covered with heat denatured digoxigenin labeled probes diluted 1/50 in hybridisation buffer (50% deionised formamide, 10% dextran sulphate, 1 mg/ml tRNA, 1×Denhardt's solution, 1× Sait buffer). The sides were incubated at 65° C. overnight in a humidity chamber and then, washed four times for 15 min with pre warmed washing solution (1×SSC, 50% formamide, 0.1% tween-20) at 65° C., two times 30 min each with MABT buffer (100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20) at RT and covered with freshly-prepared antibody blocking solution [60% (v/v), goat immunoglobulins 20% (v/v), 2% blocking reagent (Roche)] 1 hour at RT. The sections were covered with goat anti-digoxigenin antibodies coupled to alkaline-phosphatase (Roche) (1/2000 in antibody blocking solution) and incubated 4 hours at 37° C. in a humidity chamber. The slides were washed two times 30 min each in MABT buffer, rinsed two times for 10 min at RT with NTMT buffer (100 mM Tris-HCl, ph 9.5, 100 mM NaCl, 50 mM MgCl 0.1% Tween-20). The slides were covered with alkaline phosphatase substrate solution (NBT, BCIP) containing levamisole and incubated for a few hours at RT or overnight at 4° C. humidity chamber. Reaction was stopped by washing with PBS/Tween 0.1% and the tissue was counterstained with nuclear fast red (Vector labs) and mounted. Specific sense and antisense probes were synthesized by in vitro transcription of KIAA0153 expression vector, in the presence of digoxigenin-labelled UTP.
Cells were harvested by a brief trypsin treatment, transferred to tubes containing PBS-10% fetal calf serum and counted, The cells were collected by centrifugation at 950 rpm/5 min/RT. The cells were washed twice with PBS, centrifuged and resuspended in TTL buffer 1× (50 mM Tris-HCl pH 7.4, MgCl 12.5 mM, KCl 100 mM, DTT 1 mM, glycerol 5% Rnase A 50 mg/ml). The cell suspensions were subjected to 3 cycles of freezing-thawing (liquid N2-37° C.) and centrifuged at 10000 rpm for 10 min at 4° C. We assayed triplicate samples and, typically, the amount of cell extract corresponding to 3×10 cells. Alternatively, cells were washed, scraped directly in TTL buffer and lysed as indicated above. Identical amount of cell extracts were assayed. The assay mixture was incubated at 37° C. for 1 hour and contained the following reagents in addition to the TTL buffer: 4 mM ATP, 200 mg/ml purified rabbit tubulin, 0.5 mCi of L-[U-14C] Tyrosine (434 mCi/mmol; Amersham). Final reaction volume was 50 ml. The rabbit tubulin was purified by two cycles in an in vitro polymerisation system described by Shelanski et al (11). The reaction was stopped by placing the samples in ice and immediately pipetting 50 ml-samples into 1 ml of 10% trichloroacetic acid (TCA) containing 20 mg of unlabelled tyrosine (Sigma). BSA (200 mg) was added as carrier, and the TCA precipitates were kept at 0° C. for 30 min. The TCA precipitates were collected on GFC Whatman glass fiber filters. The samples were washed three times with 5 ml of 10% TCA, dried, dissolved in Scillintation liquid cocktail (ReadySafe™ Beckman) and counted in a Beckman scillintation counter.
Statistical analyses were performed using Student t-test.
Cells were transfected with the Fast Forward Protocol according to the manufacturer's instructions (Hyperfect, Qiagen). Western blots: Cells were split into 6 well plates at 70% confluence, and transfected with siRNAs. Control GFP-siRNA (Qiagen); siRNA GADP (Dharmacon); siRNAs KIAA153 [mixture of #1 (D15633670 Dharmacon) and #2 (Dharmacon, custom)]. Cells were transfected with Hyperfect (Qiagen), and incubated for 48 hours in high serum medium (without removing reagent). Cells were scraped into TGKD, proteins were determined by the Pierce (Bradford) method, and 10 μg used for WB with PAb#2089 (KIAA0153) and GAPDH (monoclonal MAb374 Chemicon). Growth curves: DU145 cells at 6K per well (96 well plates, cell number optimised for initial growth) were transfected with Hyperfect and after 1-6 days 5 wells per condition were analysed using the MTT assay (Sigma). Cell cycle progression: At various time after transfection (48 and 72 hours), the cells were incubated in low serum (4 or 16 hours, respectively), washed twice with PBS, once with PBS+3 mM EDTA, incubated in 400 μl 0.05% trypsin followed by 2 ml complete medium, centrifuged, washed in PBS, precipitated with EtOH (PBS:EtOH 1:2 vol:vol), incubated overnight at 4° C., rehydrated in cold PBS, incubated at RT for 2 hours, centrifuged, washed twice with PBS and resuspended in PBS+1 mg per ml RNAase 1, incubated 2 hours at RT. An equal volume of propidium iodide-TritonX100 was added (0.05 mg/ml and 0.1% final concentrations, respectively). The cells were incubated overnight at 4° C. in the dark, and analysed by FACScalibur cytometer (Becton Dickinson). In general 20K cells were analysed, and cell cycle distribution was estimated with CellQuest software.
KIAA0153 is differentially expressed in head & neck squamous cell carcinoma and prostate cancer (submitted and data not shown) and codes for a 72 KDa protein with a C-terminal TTL domain (
KIAA0153 expression in benign epithelial cells was rated 1.0 on average in a 4 point scoring scale. Staining was significantly increased in tumors and there was also a clear association with the Gleason pattern, with 1.6 for Gleason patterns 2-3 scored and 1.8 for patterns 4-5 scored. Scores also increased with progression, with 2.3 for locally recurrent tumors resected after failure of androgen ablation therapy, 2.6 for lymph node metastasis and 2.3 for distant metastasis. Interestingly, expression in prostate cancer precursor PIN lesions was higher than in the local tumors, almost reaching the levels of recurrent tumors and metastasis (mean score 2.1).
The presence of a TTL like domain in KIAA0153 suggests that it may have a role in microtubule functions, such as mitosis and chromosome stability. The karyotypically stable cell line HCT-116, that is widely used to study mitotic events and chromosomal instability and has a normal mitotic checkpoint, was used. The DNA profiles of stable transfectants were analyzed by flow cytometry. Cells over expressing KIAA0153 had a distinct additional peak, corresponding to 8c DNA content (where c is the amount of DNA in a haploid HCT-116 cell) compared to controls (
A synthesis of karyotypic results is presented in following Table 2:
Analysis of metaphase spreads showed that, compared to controls, clones over-expressing KIAA0153 have higher frequencies of cells with (8c), and the loss of p53 led to an increase in the proportion of cells with 90 chromosomes and the appearance of an addition population with 131 chromosomes (12c). We extended the analysis to karyotypically unstable cells with the purpose of exacerbating this striking phenotype. HEp-2 cells were chosen because they are amenable to stable transfection and synchronisation, and have relatively low levels of endogenous KIAA0153 (see below and data not shown). Individual clones over-expressing KIAA0153 had distinct new peaks with 8c DNA content, and higher G2/M peaks consistent with the presence of overlapping diploid and tetraploid cycles (
Chromosome dynamics in living cells was followed using cells stably expressing histone H2B-GFP [9, see Methods]. Unsynchronised cultures were observed for 48 hours and mitotic events were analyzed. In cells over-expressing KIAA0153 there was a striking high incidence of aberrant mitoses compared to controls. There were metaphase plates with distinct “L” and “X” shapes indicative of aberrations in the mitotic spindle that could yield daughter cells with unequal DNA content (
Since KIAA0153 has a putative TTL domain, TTL activity in transient and stably transfected cells was studied. Using in vitro assays with cell extracts from transiently transfected cells, it was found that KIAA0153 expression did not increase overall TTL activity, in contrast to expression of human TTL (
The cellular localisation of Glu-tubulin in the stable clones was also studied. Glu-tubulin is normally preferentially located in a subset of less dynamic microtubules (14-16) as well as in centrioles and mid-bodies, and is absent from astral microtubules (16-17). Tyr-tubulin is found throughout the interphase network and the mitotic spindle. KIAA0153 overexpression did not detectably alter the cellular distribution of the evidently increased levels of Glu-tubulin (
The influence of the knock down of KIAA0153 in human prostate cancer cell lines DU145, LnCAP and 22RV1 was tested using siRNAs directed against KIAA0153.
Results obtained for human prostate cancer line DU145 are presented in
Taken together, our results show that aberrant expression of the novel gene KIAA0153 is associated with prostate cancer progression. It leads to chromosome instability that could result from: inhibition of tubulin tyrosination, increases in the number of centrioles and abnormal multipolar mitoses. Interestingly, Glu-tubulin is normally enriched in centrioles, suggesting that it may have a role in centriole function (18-19) and that KIAA0153 may perturb this function. Furthermore, our preliminary data suggests that KIAA0153 interacts with a centriolar protein (data not shown). KIAA0153 synergises with loss of p53 in the induction of chromosome instability, consistent with it having oncogenic properties (13). KIAA0153 may favour tumorigenesis by increasing chromosome instability, or alternatively by affecting the incorporation of endogenous nitrotyrosine into α-tubulin. Nitrotyrosination of alpha-tubulin in microtubules may act as a “last check point” by triggering apoptosis in aberrant cells (20,21). Expression analysis in benign and malignant prostate tissue revealed a significant increase of expression in tumors and association of expression levels with the progression steps of prostate cancer. Immunoreactivity in tumor specimens increases from untreated primary tumors to local recurrent, androgen-insensitive tumors and metastases. Within the group of primary tumors expression levels were higher in the Gleason pattern 4 and 5 tumor regions as compared to patterns 2 and 3. The presence of the two highest Gleason patterns in a tumor significantly increase the risk of progression suggesting that KIAA0153 levels at the time of initial prostate cancer treatment are related to the risk of progression and requirement of more aggressive treatment. Interestingly, expression in PIN lesion, which are considered prostate cancer precursor lesions, were almost as high as in recurrent disease and metastases, suggesting that upregulation of KIAA0153 expression is an early event of malignant changes occurring in the prostate gland. Highest levels in tumors progressing after escape from androgen ablation therapy and much higher expression in tumors as compared to benign prostate tissue identify the KIAA0153 as a potential target for therapy of prostate cancer after failure of androgen ablation treatment.
Finally, promising results have been obtained by knocking down KIAA0153 expression in human prostate cancer cell lines using siRNA directed against KIAA0153, thus demonstrating the interest of KIAA0153 as a target for prostate cancer therapy.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/050761 | 1/23/2008 | WO | 00 | 7/22/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60881830 | Jan 2007 | US |
